The present invention relates to a method of identifying peptides of a desired origin and which are capable of binding to MHC molecules of a particular haplotype; peptides identified by the method; pharmaceutical compositions containing the peptides, databases describing the peptides and the use of the peptides in vaccination.
The following abbreviations are used herein: MHC, Major Histocompatibility Complex; β2 m, β2-microglobulin; ESI, electrospray ionization; MS, mass spectrometry; m/z, mass charge ratio; CID, collision induced disintegration; MS/MS, tandem mass spectrometry; MTDM, DNA methyl transferase; FAS, fatty acid synthase; CTL, cytotoxic T lymphocytes; mAbs, monoclonal antibodies.
The MHC serves as a shuttle to transport and display peptide antigens on the surface of cells as an indication to the immune system of the health state of the cells. Each individual has at most six different MHC class-I haplotypes, out of the hundreds known. MHC bound peptides, i.e., peptides bound to, and presented in context of, MHC molecules, originate from proteolysis of most of the proteins expressed in the cells. Therefore, unique sets of MHC bound peptides are displayed by each of the different MHC haplotypes according to the protein expression and degradation schemes of the cells and according to the peptide binding motifs of the MHC molecules (reviewed in [1]). Therefore, thousands of different peptides are presented by the different MHC class-I haplotypes and each of the peptides is presented in vastly differing copies per cell [2]. When cells become infected, some of the presented peptides are derived from the pathogen's proteins, and so indicate to circulating T-cells to kill the diseased cells and prevent the spread of the disease.
Each MHC haplotype recognizes the peptides through a broadly defined consensus motif of peptide's amino acids strategically positioned to serve as anchors to the appropriate binding pockets on the MHC molecule. The binding motifs of many of the MHCs haplotypes were first established by pool Edman sequencing of unfractionated peptide mixtures eluted from immunoaffinity purified MHC molecules [3, 4]. The consensus was further extended by direct biochemical analysis of individual peptides separated by reversed phase chromatography and analyzed by tandem mass spectrometry [2, 5, 6], reviewed in [7].
MHC bound peptides derived from cancer specific or associated proteins or antigens were extensively searched for, with the goal of finding among them peptide candidates for development of anti-cancer vaccines. A number of such tumor specific peptides were already identified and some were successfully tested as anti-cancer vaccines for human treatment, most notably for immunotherapy of melanoma [8, 9]. Three main approaches were extensively used for the identification of such MHC bound peptides [10]. The genetic approach involves transfection of cDNA libraries, made from tumor cells, into cells that present the MHC allele of interest. The clones of transfected cells that stimulated CTL lines against the tumor cells were selected as the source for the tumor antigen and the genes were further fragmented to isolate the regions of the genes that encode the particular immunogenic peptide [11]. The second approach is based on exploiting the known consensus binding motifs of the MHC haplotype of interest to scan sequences of known protein “in silico” and to predict putative MHC bound peptides that fit this consensus [12]. For successful prediction, these consensus motifs should be a prior well established, which is not the case for many of the MHC haplotypes [13]. The drawback of this approach is its reliance on chemical synthesis of a large number of peptides, only few of which end up being useful. The biochemical, third approach, involves the fractionation of the MHC bound peptides by chromatography, assaying the fractions for immuonoglogical activity and sequencing the individual peptides in the active fractions [2, 5]. The biochemical approach is the only possible way to identify post-translationally modified peptides, not always predictable from the protein sequences [14-16]. The biochemical approach depends on the availability of advanced mass spectrometry, needed for analyzing the available minute amounts of peptides that are present at very complex mixtures (reviewed in [7]).
All these approaches for identifying MHC bound peptides eventually rely on chemical synthesis of the peptides of interest to test their capacity to bind to the MHC molecule by stabilization of empty MHC molecules on cell surface [17], and their potential to elicit an immune response by tetramer assays [18], ELISPOT [19]and elicitation CTL responses when presented on cells [20].
Currently, sequencing and identification of individual MHC bound peptides the direct biochemical approach is most effectively performed by use of tandem mass spectrometry. The peptides are resolved by reversed phase chromatography and the elating peptides are collected, assayed for biological activity and sequenced, most often by electrospray tandem mass spectrometry [2, 5, 21]. Comparing the patterns of MHC bound peptides recovered from healthy and infected cells helps to identify disease related peptides [22]. Mass spectrometry is advantageous for such analysis due to its accuracy speed of analysis, its ability to analyze complex mixtures of peptides and its high sensitivity [7]. The biochemical analysis involves the purification of the MHC molecules with their bound peptides by immunoaffinity chromatography using mAbs specific for the native MHC [2]. To this end, the cells are solubilized with detergents, the desired MHC molecules are purified with their MHC bound peptides and the MHC bound peptides are recovered by denaturation and ultra-filtration. However, once the cells are disrupted by the detergents, the MHC molecules become contaminated by cellular debris and detergents which complicates the subsequent ESI-MS/MS analysis. Moreover, such immunoaffinity purification of desired MHC haplotypes is possible only when specific mAbs are available, whereas for many MHC haplotypes such mAbs are presently unavailable.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method for identifying MHC bound peptides devoid of the above limitations.